Protein-mediated membrane fusion is essential for maintaining eukaryotic cell organization and propagation of many clinically relevant human viruses. Members of the paramyxovirus family typically rely on the concerted action of two envelope glycoproteins, the attachment and fusion protein, to fuse their envelope with the target cell plasma membrane for cell entry. However, basic questions concerning fundamental mechanistic principles of the paramyxovirus entry machinery are not addressed: What is the spatial organization of the envelope glycoprotein hetero-oligomer complexes in the native, metastable conformation? How does receptor binding affect attachment protein organization? What is the nature of the intermolecular contacts that link receptor binding with coordinated refolding into the thermodynamically stable postfusion conformation? With crystal structures of isolated ectodomains of different paramyxovirus glycoproteins at hand, this project will address these questions by pairing innovative imaging technology with biochemical, functional and computational approaches. Cryo-electron tomography will elucidate the organization of hydrated, native fusion complexes displayed on the surface of viral particles, alone and complexed with soluble receptor molecules (aim 1). Native gel electrophoresis will parallel the electron tomography approach and elucidate the molecular nature of the signal that links receptor binding to F refolding (aim 2). Molecular modeling, directed mutagenesis and peptide interference will expand, characterize and confirm candidate short-range intermolecular contacts that pilot studies have identified as instrumental for the formation of functional fusion complexes (aim 3).